identification of potential saliva and tear biomarkers in primary sj gren s syndrome utilising the extraction of extracellular vesicles and proteomics analysis

A small volume from the tear fluid sample of each participant 100 μl, the joint fractions from each individual, and the joint fractions from pooled tear samples of the patients with pSS

R E S E A R C H A R T I C L E Open Access

Identification of potential saliva and tear

utilising the extraction of extracellular

vesicles and proteomics analysis

Lara A Aqrawi1* , Hilde Kanli Galtung2, Beate Vestad3, Reidun Øvstebø3, Bernd Thiede4, Shermin Rusthen1, Alix Young5, Eduarda M Guerreiro2, Tor Paaske Utheim2,3,6, Xiangjun Chen6, Øygunn Aass Utheim3,7,

Øyvind Palm8and Janicke Liaaen Jensen1

Abstract

Background: There is a long-lasting need for non-invasive, more accurate diagnostic techniques when evaluating primary Sjögren’s syndrome (pSS) patients Incorporation of additional diagnostics involving screening for

disease-specific biomarkers in biological fluid is a promising concept that requires further investigation

In the current study we aimed to explore novel disease biomarkers in saliva and tears from pSS patients

Methods: Liquid chromatography-mass spectrometry (LC-MS) was performed on stimulated whole saliva and tears from 27 pSS patients and 32 healthy controls, and salivary and tear proteomic biomarker profiles were generated LC-MS was also combined with size exclusion chromatography to isolate extracellular vesicles (EVs) from both fluids Nanoparticle tracking analysis was conducted on joint fractions from the saliva and tears to determine size

distribution and concentration of EVs Further EV characterisation was performed by immunoaffinity capture of CD9-positive EVs using magnetic beads, detected by flow cytometry The LC-MS data were analysed for quantitative differences between patient and control groups using Scaffold, and the proteins were further analysed using the Database for Annotation, Visualization and Integrated Discovery (DAVID), for gene ontology overrepresentation, and the Search Tool for the Retrieval of Interacting Genes/Proteins for protein-protein interaction network analysis Results: Upregulation of proteins involved in innate immunity (LCN2), cell signalling (CALM) and wound repair (GRN and CALML5) were detected in saliva in pSS Saliva EVs also displayed biomarkers critical for activation of the innate immune system (SIRPA and LSP1) and adipocyte differentiation (APMAP) Tear analysis indicated

overexpression of proteins involved in TNF-α signalling (CPNE1) and B cell survival (PRDX3) Moreover, neutrophil gelatinase-associated lipocalin was upregulated in saliva and tears in pSS Consistently, DAVID analysis

demonstrated pathways of the adaptive immune response in saliva, of cellular component assembly for saliva EVs, and of metabolism and protein folding in tears in pSS patients

Conclusions: LC-MS of saliva and tears from pSS patients, solely and in combination with size-exclusion chromatography allowed screening for possible novel biomarkers encompassing both salivary and lacrimal disease target organs This approach could provide additional diagnostic accuracy in pSS, and could possibly also be applied for staging and

monitoring the disease

Keywords: Sjögren’s syndrome, Autoimmunity, Inflammation, Innate immunity, Adaptive immunity, Saliva, Tears,

Proteomics, Extracellular vesicles, Biomarkers

* Correspondence: l.a.aqrawi@odont.uio.no

1 Department of Oral Surgery and Oral Medicine, Faculty of Dentistry,

University of Oslo, Oslo, Norway

Full list of author information is available at the end of the article

© The Author(s) 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made The Creative Commons Public Domain Dedication waiver

Sjögren’s syndrome (SS) is a systemic rheumatic

auto-immune disease, where chronic inflammation results in

progressive destruction of exocrine glands, primarily the

lacrimal and salivary glands [1, 2] Thus, characteristic

features are sicca symptoms, including dry eyes and dry

mouth [3] The prevalence of SS has been reported to be

between 0.01% and 0.6% [4–6]

The main classification criteria used today when

diagnos-ing primary SS (pSS) are the American-European

Consen-sus Group (AECG) criteria from 2002 [7], which rely on

evaluating symptoms of ocular and oral dryness, assessing

the secretory ability of the exocrine glands, screening for

anti-Ro and anti-La autoantibodies, and evaluating biopsies

of minor salivary glands for mononuclear cell infiltration

[8] This routine assessment of minor salivary gland tissue

and histological focus scoring has been employed to

describe salivary gland involvement in SS [9, 10] Here, a

positive biopsy with mononuclear cell infiltrates

compris-ing≥50 mononuclear cells per 4 mm2

resulted in a positive focus score value ranging from 1 to 12 according to the

number of foci seen This is a semi-quantitative, invasive

technique useful for patients with glandular dysfunctions

without autoantibody production [11]

Considering the nature of the currently available

diagnos-tic tools, there remains an unmet need for non-invasive,

more accurate diagnosis of pSS The incorporation of

additional non-invasive diagnostics, such as screening for

disease-specific biomarkers [12, 13] has therefore been in

focus over recent decades, as it can also be applied for

staging and monitoring of the disease Indeed, liquid

chromatography-mass spectrometry (LC-MS) has been

applied in several human rheumatic diseases, including SS,

in order to discover biomarkers and therapeutic targets by

studying the proteome of biological fluids [14, 15] Both

sal-iva [14, 16–21] and tear fluid [22, 23] have previously been

used to identify potential biomarkers for SS It has been

re-ported that oral fluid not only reflects the salivary gland

in-volvement that characterises SS disease [18, 24, 25], but also

has the potential to represent the subject’s current general

health [26, 27] Moreover, salivary fluid samples can easily

be obtained using a non-invasive, simple, safe, and

stress-free procedure, allowing for repetition and multiple

collec-tions This explains why the majority of proteomic studies

of SS have chosen saliva as the ideal biological fluid,

exam-ining either whole saliva or saliva from individual glands

(e.g minor and/or parotid salivary glands), under both

stimulated and unstimulated conditions [14, 16–21] As a

result, several common biomarkers for SS have been found,

including secretory proteins, enzymes, highly abundant

immune-system-related molecules (e.g β2-microglobulin),

and cytokines such as IL-4 and IL-5 [21, 28, 29]

Proteomic analyses can also be coupled with various

separ-ation techniques in order to isolate the cellular components

of interest when screening for disease biomarkers Extracel-lular vesicles (EVs) are an example of such celExtracel-lular compo-nents These are membrane-embedded vesicles, comprising exosomes (size <100 nm) and/or microvesicles (size 100–

1000 nm) [30], released by cells that are emerging as import-ant mediators of intercellular communication, and thereby influencing recipient cell functions [31–33] For instance, EVs can act on the innate immune system as paracrine messengers and have been described as pro-inflammatory mediators that induce inflammatory signals during infections [34, 35] and chronic inflammatory diseases [35]

Interestingly, patients with autoimmune diseases have increased levels of EVs that carry components associated with complement activation [36, 37] Accordingly, various cell types of the innate immune system are known to release EVs, including macrophages [38], monocytes or dendritic cells [39] and natural killer (NK) cells [40] Besides mediat-ing the exchange of intercellular information by their surface molecules, EVs have been shown to be carriers of important soluble mediators, such as cytokines The involvement of EVs in the transport of the cytokines IL-1b [41] and tumour necrosis factor (TNF) [42] are such examples

Proteomic profiling of EVs in a biological context can be challenging, especially if the EV preparations are not highly purified [43] In complex body fluids, EVs can be separated from interfering molecules, such as proteins and lipids, by utilising size-exclusion chromatography [44, 45] The isolated sub-fractions containing the highest EV concentra-tions can then be characterised using nanoparticle tracking analysis, and by flow cytometry detection of the fusogenic protein/tetraspanin CD9, which is abundantly expressed in EVs [46–48]

Proteomic studies of isolated EVs have in turn yielded extensive catalogues that display which proteins are abundant in different types of EVs, specifically reflecting vesicle localization, cellular origin, and mechanism of secre-tion [49] Hence, in the current study we hypothesised that

by applying LC-MS alone, and in combination with EV-isolation, using samples of stimulated whole saliva and tear fluid from patients with pSS and healthy controls, novel biomarkers may be identified encompassing both salivary and lacrimal disease target organs Such biomarkers may in turn be implemented, as potential non-invasive diagnostic tools that can help to increase diagnostic accuracy when evaluating patients with pSS, in accordance with the AECG criteria, and can also be useful when monitoring disease progression

Methods

Study population

Patients with pSS (n = 27) that fulfilled the AECG classifica-tion criteria from 2002 [7] and 32 age-matched and gender-matched controls participated in this study Following recruitment at the Department of Rheumatology, Oslo

University Hospital, the patients were referred to the Dry

Mouth Clinic, located at the Institute of Clinical Dentistry,

Faculty of Dentistry, University of Oslo, and the Norwegian

Dry Eye Clinic, Oslo, for thorough examination and sample

collection, as described below A detailed explanation of the

study aim and protocols were explained to the recruited

subjects upon enrolment Written informed consent was

obtained from the participants and the Regional Medical

Ethical Committee of South-East Norway approved the

study (2015/363)

Medical records and clinical data were obtained through

clinical examination and from patients’ charts at the

De-partment of Rheumatology, Oslo University Hospital This

provided information that had been collected during

rou-tine laboratory assessments, including anti-Ro/SSA and

anti-La/SSB, and evaluation of ocular and oral dryness by

assessing saliva and tear secretion Some residual secretory

ability was required for inclusion of the patients in the

study The demographic data for the patients included in

this study are presented in Table 1

Saliva and tear fluid collection

Saliva collection at the Dry Mouth Clinic

Participants underwent a thorough oral examination at the

Dry Mouth Clinic, and stimulated whole saliva was

col-lected from all participants Subjects were asked to not have

any food or drink for at least 1 hour before saliva collection

Following the oral examination, the participants were asked

to chew on a paraffin block (Paraffin Pellets, Ivoclor

Viva-dent, Shaen, Lichtenstein), while saliva was collected on ice

for 5 minutes between 9.00 a.m and 3.00 p.m As secretory

ability has been shown to vary depending on stimulation by

chewing, and on the time of day, these strict routines were

employed to ensure standardisation of the method for saliva

collection The samples were weighed to determine volume,

where only patients producing≥800 μl of stimulated whole

saliva were included in the study All samples were then

ali-quoted and stored at -80 °C

Tear fluid collection at the Norwegian Dry Eye Clinic

Participants underwent a thorough ocular surface

exam-ination at the Norwegian Dry Eye Clinic Tear fluid was

collected from both eyes by placing a Schirmer tear test

strip (HAAG-STREIT, Essex, UK) on each eye for 5

mi-nutes, or more to produce a minimum combined total

of 10 mm of tear volume from both eyes at room

temperature Each Schirmer strip was then transferred

to 500 μl of 0.1 μm filtered phosphate-buffered saline

(PBS) (Gibco, pH 7.4, ThermoFisher Scientific, Oslo,

Norway) and stored at -80 °C

Extraction of EVs from saliva

EVs were isolated from stimulated whole saliva using

size-exclusion chromatography, as described previously

[44] In brief, the saliva samples were centrifuged at

300 rpm for 10 minutes to remove debris, and then di-luted 1:2 with 0.1μm filtered PBS A qEV size-exclusion chromatography column (iZON Science, Oxford, UK) was equilibrated by washing the column with 15 ml of 0.1 μm filtered PBS; 1 ml of the diluted saliva was then applied to the column and 16 fractions, each 500 μl in volume, were collected by continuously adding 0.1 μm filtered PBS to the column To standardise the proced-ure, elution time frames were recorded when reaching fractions 7, 12 and 15, and the number of eluted drops

in fraction 10 was also recorded A new column was used for each saliva sample The eluted fractions 8− 10 (containing the majority of microvesicles and exosomes

Table 1 Clinical characteristics of patients with pSS included in the study

ID number

Age (years)

Anti-SSAb

Anti-SSBb

Schirmer testc

Saliva secretiond

Dry mouth

Dry eyes

pSS21 a

a

Patients with pSS included in pooled tear sample only b

Autoantibody production was assessed by ELISA c

Values are in mm/5 minutes; normal flow

>5 mm/5 minutes The + symbol indicates dryness and tear secretion <5 mm/

5 minutes.dValues are in ml/15 minutes; normal flow >1.5 ml/15 The + symbol indicates dryness and stimulated whole saliva secretion <3.5 ml/5 minutes

present in the samples) were concentrated for 80 minutes

at 30 °C in a MiVac centrifugal vacuum concentrator (SP

Scientific, Suffolk, UK) from a volume of 500 μl to

approximately 250μl Fractions 8–10 were collected into

a joint fraction and the protein concentration was

deter-mined using Qubit Fluorometric Quantitation

(Thermo-Fisher Scientific, Oslo, Norway) A volume of the diluted

stimulated whole saliva (100 μl) and the joint fractions

from each participant were then sent for proteomic

ana-lysis while preserved on dry ice

Extraction of EVs from tear fluid

For each subject, tear fluid eluted from Schirmer strips into

0.1μm filtered PBS (1 ml; pooling of 500 μl PBS containing

a Schirmer strip from each eye) was applied to an

equili-brated qEV size exclusion chromatography column

Frac-tions of 500μl were eluted and concentrated, and fractions

8–10 were collected into a joint fraction and the protein

concentration was determined as described above A new

column was used for each sample Due to the low numbers

of proteins and vesicles in tear fluid collected from the

indi-vidual patients with pSS (minimum 10 mm fluid per

pa-tient), tear fluid from Schirmer strips containing 80 mm

tear fluid from 11 patients with pSS was pooled in 5 ml

PBS The pooled sample was subsequently concentrated to

200 μl using Amicon Ultra-4 columns and furthermore

adjusted to a volume 1.0 ml with PBS before being applied

on a qEV column Schirmer strips also containing 80 mm

tear fluid from five controls were handled in parallel These

pooled tear fluid samples were included for verification A

small volume from the tear fluid sample of each participant

(100 μl), the joint fractions from each individual, and the

joint fractions from pooled tear samples of the patients with

pSS and the controls were then sent for proteomic analysis

while preserved on dry ice

Characterisation of EVs

Nanoparticle tracking analysis

Nanoparticle tracking analysis was conducted on joint

fractions from saliva and tear fluid to determine size

distribution and concentration of the respective EVs using

a NanoSight NS500 instrument (Malvern Instruments

Ltd, Malvern, UK), equipped with a scientific cMOS

camera with trigger, a 488-nm laser, and a syringe pump

for continuous sample flow Samples were diluted in

0.02 μm filtered PBS to reach the measurement range

(108− 109

particles/ml) Analysis was performed using the

NTA 3.0 software (Malvern Instruments, Malvern, UK)

Briefly, a video capture of 60 seconds per sample was

ap-plied The camera level was set to 14–15 for saliva and

12–15 for tear fractions, and the detection threshold was

set to 3 The hydrodynamic diameter of the particles in

each sample was calculated by the software, through

regis-tering their Brownian motion in response to laser light

scattering, utilising the Stokes Einstein equation Sample concentration was estimated as a subsequent parameter of the sample volume A summary of the measurements ob-tained from the nanoparticle tracking analysis for EV char-acterisation in saliva and tear fluid is presented in Table 2

Flow cytometry detection of CD9 positive EVs

Immunoaffinity capture and detection of CD9 positive EVs from joint fractions was performed using the Exo-some Human CD9 Flow Detection Kit (Dynal®, Thermo-Fisher Scientific, Oslo, Norway) and flow cytometry In brief, 100μl of each joint fraction was incubated overnight with prewashed 20 μl Dynabeads (2.7 mm) on a Hula-Mixer Sample mixer at 4 °C The bead-captured EVs were then washed three times with 0.1μm filtered PBS contain-ing 0.1% bovine serum albumin (BSA) Subsequently, they were incubated with RPE-conjugated detection antibody (anti-human CD9-RPE clone ML-13, BD Biosciences, Oslo, Norway), or isotype control (IgG1-RPE, BD Biosci-ences, Oslo, Norway), for 45 minutes at room temperature

on an orbital shaker (1000 rpm), protected from light The bead-containing samples were further washed twice with PBS containing 0.1% BSA before proceeding with flow cytometry analysis, using a BD Accuri™ C6 Cytometer (BD Biosciences, Oslo, Norway) Median fluorescence intensity (MFI) was reported as a signal to noise (S/N) ratio to isotype control from a total of 300 singlet events Measurements obtained from the flow cytometry analyses

Table 2 Characterisation of EVs in saliva and tear fluid

Mean particle size a (nm)

Particles/ml a CD9+ EVs

S/N ratio MFI b

Saliva Patients with pSS

189 ± 4.1 5.46 E + 10 ± 1.43 E + 10* 3.47 ± 0.56* Controls 189 ± 4.4 2.41 E + 10 ± 3.98 E + 09 1.93 ± 0.15 Tear fluid

Patients with pSS

171 ± 6.9 1.54 E + 09 ± 3.08 E + 08 1.10 ± 0.03 Controls 163 ± 9.6 1.09 E + 09 ± 1.06 E + 08 1.06 ± 0.02 Pool of

patients with pSS

Pool of controls

a

Nanoparticle tracking analysis was conducted on extracellular vesicles (EV) joint fractions from whole saliva (n = 19 patients with primary Sjögren ’s syndrome (pSS), n = 32 controls), tear fluid (n = 7 patients with pSS, n = 6 controls), and one pooled tear sample (n = 11 patients with pSS, n = 5 controls)

to determine mean particle size of microvesicles and exosomes (nm ± SEM), in addition to concentrations of EVs (particles/ml ± SEM) b

Detection of CD9+ EVs from joint fractions of saliva (n = 19 patients with pSS, n = 32 controls), tear fluid (n = 11 patients with pSS, n = 10 controls), and one pooled tear sample (n = 11 patients with pSS, n = 5 controls) was performed by immunoaffinity capture using anti-CD9-coated magnetic beads followed by flow cytometry analysis The results were reported as signal-to-noise (S/N) ratios of median fluorescence intensity (MFI) *Significant difference between patients with pSS and controls (unpaired t test, p < 0.05)

for EV characterisation in saliva and tear fluid are

pre-sented in Table 2

Determination of protein amount

Proteomics analysis was executed on saliva and tear fluid

from both patients with pSS and controls before and after

isolation of EVs Total protein concentration (mg/ml) in

the saliva samples ranged from 0.5 to 1.36 in patients with

pSS, and from 0.25 to 0.94 in controls Meanwhile, saliva

joint fractions showed a total protein range of 0.04 to 0.07

in patients with pSS, and 0.02 to 0.07 in controls The total

protein concentration in tear samples ranged from 0.27 to

0.70 in patients with pSS, while in controls this ranged from

0.22 to 0.70 Tear joint fractions displayed a total protein

range from 0.03 to 0.05 in patients with pSS, and 0.03 to

0.04 in controls The total protein in the pooled tear sample

was 0.47 mg/ml in the patients with pSS and 0.35 mg/ml in

the controls Additionally, the joint fractions in the pooled

tear sample exhibited a total protein value of 0.03 mg/ml in

the patients with pSS, and 0.03 mg/ml in the controls

In-solution protein digestion

For saliva and EVs of saliva, four times the sample volume

of ice-cold acetone was added to each sample, vortexed and

precipitated overnight at -20 °C Samples were then

centri-fuged at 16,000 g for 20 minutes at 4 °C (Centrifuge 5415R,

Eppendorf, Hamburg, Germany) and the supernatants were

discarded Proteins were re-dissolved in 50μl of a mixture

of 6 M urea and 100 mM ammonium bicarbonate (pH 7.8)

For reduction and alkylation of cysteines, 2.5μl of 200 mM

DTT in 100 mM Tris-HCl (pH 8) was added and the

sam-ples were incubated at 37 °C for 1 hour followed by the

addition of 7.5μl of 200 mM iodoacetamide for 1 hour at

room temperature in the dark The alkylation reaction was

quenched by adding 10 μl of 200 mM DTT at 37 °C for

1 hour For all samples, the proteins were digested with

10 μg of trypsin for 16 hours at 37 °C The digestion was

stopped by adding 5μl of 50% formic acid The generated

peptides were purified using an OMIX C18-micro SPE

(Agilent, Santa Clara, CA, USA), and then dried using a

Speed Vac concentrator (Concentrator Plus, Eppendorf,

Hamburg, Germany)

Liquid LC-MS

The tryptic peptides were dissolved in 10μl of 0.1% formic

acid/2% acetonitrile, and 5 mμl was analysed using an

Ultimate 3000 RSLCnano-UHPLC system connected to a Q

Exactive mass spectrometer (Thermo Fisher Scientific,

Bre-men, Germany), and also equipped with a nano electrospray

ion source For liquid chromatography separation, an

Ac-claim PepMap 100 column was used (C18, 2 μm beads,

100 Å, 75 μm inner diameter, 50 cm length) (Dionex,

Sunnyvale CA, USA) A flow rate of 300 nl/minute was

employed with a solvent gradient of 4− 35% B in 47 minutes,

to 50% B in 3 minutes and then to 80% B in 2 minutes Solvent A was 0.1% formic acid and solvent B was 0.1% formic acid/90% acetonitrile

The mass spectrometer was operated in the data-dependent mode to automatically switch between MS and MS/MS acquisition Survey full-scan MS spectra (from m/z

300 to 2,000) were acquired with the resolution R = 70 000

at m/z 200, after accumulation to a target of 1e6 The max-imum allowed ion accumulation time was 60 milliseconds The method used allowed sequential isolation of up to the ten most intense ions, depending on signal intensity (inten-sity threshold 1.7e4), for fragmentation using higher-energy collisional-induced dissociation (HCD) at a target value of 10,000 charges, and a resolution R = 17,500 Target ions already selected for MS/MS were dynamically excluded for

60 seconds The isolation window was m/z = 2 without off-set For accurate mass measurements, the lock mass option was enabled in MS mode

Data were acquired using Xcalibur v2.5.5 and raw files were processed to generate peak list in Mascot generic format (*.mgf ) using ProteoWizard release version 3.0.331 Database searches were performed using Mascot in-house version 2.4.0 to search the SwissProt database (Human, 20,279 proteins) assuming the digestion enzyme trypsin, at maximum of one missed cleavage site, fragment ion mass tolerance of 0.05 Da, parent ion tolerance of

10 ppm, and oxidation of methionines, and acetylation of the protein N-terminus as variable modifications For saliva and EVs of saliva, carbamidomethylation of cyste-ines as fixed modification was used in addition

Data processing and statistical analysis

Scaffold (version Scaffold_4.4, Proteome Software Inc., Portland, OR, USA) was used to validate MS/MS-based peptide and protein identifications Peptide identifications were accepted if they could be established at greater than 95.0% probability by the Scaffold Local false discovery rate (FDR) algorithm Protein identifications were accepted if they could be established at greater than 99.0% probability For label-free quantification, the entire MS2 total ion current (TIC) across all biological replicates was evaluated using the t test For functional analysis of the proteomics data, the Search Tool for the Retrieval of Interacting Genes/ Proteins) (STRING) (http://string-db.org/) and the Database for Annotation, Visualization and Integrated Discovery (DAVID) (v 6.7, https://david.ncifcrf.gov) were applied Stim-ulated whole saliva, saliva EVs (joint fractions) and tear fluid were analysed individually, comparing the 10 patients with pSS and controls with the highest number of proteins STRING was used to explore how these proteins were inter-related to form protein-protein interaction networks, by applying all active interaction sources (experiments, data-bases and text mining), and medium confidence Further-more, DAVID was applied, using an FDR with a maximum

5% cutoff, in order to delineate specific cellular pathways

in-volving these upregulated proteins in the patients with pSS

The unregulated group of proteins was also examined and

compared to the DAVID analysis for each of stimulated

whole saliva, saliva EVs (joint fractions) and tear samples

Results

Workflow for the identification of proteins upregulated in

patients with pSS

The proteome of saliva, tear fluid, and EVs of both saliva

and tear fluid from patients and controls were examined

by digestion of the proteins with trypsin, analysis of the

proteins by LC-MS, identification of the proteins using

Mascot database searches and further data analysis using

Scaffold to find quantitative differences based on thet test

applied on MS2 total ion currents Significantly

upregu-lated proteins withp values <0.05 according to the t test

were further analysed using DAVID for gene ontology

(GO) term overrepresentation and STRING for

protein-protein network analysis

Upregulation of proteins involved in innate immunity, cell

signalling and wound repair in whole saliva from patients

with pSS

LC-MS analysis of whole saliva from 20 patients and 32

healthy controls identified approximately 500 unique

pro-teins with 48,424 peptide spectrum matches Thirty-eight

proteins were upregulated in whole saliva in the pSS patient

group compared to the controls (Scaffold: t test, p < 0.05)

(Additional file 1: Table S1)

GO overrepresentation analysis using DAVID indicated

that cellular pathways for the upregulated proteins in the

whole saliva from the pSS patient group, in comparison to

unregulated proteins, included lymphocyte-mediated

im-munity, calcium ion binding and the neutrophin signalling

pathway These pathways are all components of the

adap-tive immune response (Fig 1)

The STRING analysis revealed that the upregulated

proteins in whole saliva from patients with pSS formed two

distinct proteprotein interaction networks; one is

in-volved in metabolism and redox reactions, while the other

plays a central role in both innate and adaptive immunity

(Fig 2) Assuming proteins found only in the pSS patient

group would be the most promising candidates for

poten-tial disease biomarkers, we also considered the number of

biological replicates in our analyses Accordingly, the five

most deviating upregulated proteins considering biological

replicates and spectral counts were neutrophil

gelatinase-associated lipocalin (LCN2), granulins (GRN), calmodulin

(CALM), epididymal secretory protein 1 (NPC2), and

calmodulin-like protein 5 (CALML5), in descending order

(Table 3, Additional file 2: Figure S1) The most upregulated

protein in whole saliva from patients with pSS, namely

LCN2, is an iron-binding protein involved in apoptosis and

the innate immune system, and is particularly responsible for the activation of neutrophils It is also an indicator of acute renal failure LCN2 is therefore present within the protein network involved in both innate and adaptive im-munity (Fig 2) GRN is a cytokine-like peptide that is cen-tral in inflammation due to its active role in wound repair and tissue remodelling CALM and CALML5 are calcium-binding proteins and play a role in intracellular signalling and differentiation of keratinocytes, respectively ESP1 is a cholesterol transporter involved in cholesterol homeostasis within the endosome and/or lysosome (Table 3)

EVs in whole saliva from patients with pSS express abundant proteins vital for activation of the innate immune system and adipocyte differentiation

LC-MS analysis of EVs from whole saliva from 20 patients and 32 healthy controls identified around 500 unique teins with 48,620 peptide spectrum matches Thirty six pro-teins were significantly upregulated in patients with pSS compared to controls (Scaffold,t test, p < 0.05) (Additional file 1: Table S2)

The DAVID analysis of EVs from whole saliva revealed cellular pathways involved in adhesion, cytoskeleton or-ganisation and membrane fusion Together, these path-ways are involved in cellular component assembly, and possess the most significantly changed GO terms when compared with the identified unregulated proteins (Fig 1) One major protein-protein interacting network was iden-tified for the upregulated proteins in EVs isolated from whole saliva from patients with pSS This observation was visualised using STRING analysis (Fig 3) These proteins are active players in the cytoskeleton, and are also involved

in cell migration and cell junction The five upregulated proteins that deviated most in biological replicates between patients with pSS and controls and that were detected in EVs from whole saliva were adipocyte plasma membrane-associated protein (APMAP), guanine nucleotide-binding protein subunit alpha-13 (GNA13), WD repeat-containing protein 1 (WDR1), tyrosine-protein phosphatase non-receptor type substrate 1 (SIRPA), and lymphocyte-specific protein 1 (LSP1) (Table 4, Additional file 3: Figure S2) The most changed of these proteins in EVs from whole saliva, APMAP, is an enzyme central in adipocyte differenti-ation Moreover, GNA13 is a G-protein that consequently plays a role in transmembrane signalling, while WDR1 is a regulatory protein involved in the disassembly of actin filaments Interestingly, SIRPA is a glycoprotein present in innate immunity, particularly in the regulation of NK cells and dendritic cell inhibition LSP1 is an actin-binding pro-tein also involved in innate immunity, specifically neutro-phil activation, and chemotaxis (Table 4) Out of the five most upregulated proteins; both GNA13 and WDR1 are present within the protein network identified (Fig 3)

Overexpression of proteins involved in TNF-α signalling

and B cell survival detected in tear fluid from patients

with pSS

Tear fluid from 11 patients and 11 healthy controls was

analysed using LC-MS, and more than 900 unique

pro-teins were identified with 75,701 peptide spectrum

matches The application of MS2 TICs using Scaffold,

following proteomic analysis, allowed the identification

of 197 significantly upregulated proteins in tear fluid

from the patient group (Additional file 1: Table S3)

DAVID revealed cellular pathways distinguished from

up-regulated proteins in tear fluid from patients with pSS,

which entail the Pentose phosphate pathway, the

tricarb-oxylic acid/Krebs cycle, and oxidoreductase activity, which

are all elements of metabolism and protein folding (Fig 1)

By applying STRING analysis we were able to visualise

two protein-protein interaction networks encompassing the

upregulated proteins in tear fluid from patients with pSS;

one is involved in redox-reactions and oxidative stress,

while the other protein-protein network is central in the

formation of the cytoskeleton and cell migration (Fig 4)

The five most upregulated proteins present in tear fluid

from patients with pSS were DNA (apurinic or apyrimidinic

site) lyase (APEX1), thioredoxin-dependent peroxidase

re-ductase (PRDX3), copine (CPNE1), aconitate hydratase

(ACO2), and LIM domain only protein 7 (LMO7) (Table 5,

Additional file 4: Figure S3) Interestingly, APEX1 is an

en-zyme that is activated in response to oxidative stress, and is

involved in DNA repair and the regulation of

transcrip-tional factors PRDX3 is an enzyme that regulates

NF-kappa-B activation, and thereby plays a central role in B cell

survival CPNE1 is a calcium-dependent

phospholipid-binding protein involved in TNF-α receptor signalling, and

in turn in inflammation and apoptosis ACO2 is an enzyme

of the Krebs cycle with a key role in carbohydrate metabol-ism Finally, LMO7 is described as a multifunctional pro-tein with a central role in cell signalling, cell adhesion, and ubiquitination (Table 5) Of the five most deviating upregu-lated proteins in patients with pSS, PRDX3 is present within the protein network involving redox-reactions and oxidative stress (Fig 4)

Additionally, a combined analysis of protein changes

in both stimulated whole saliva and tear fluid was per-formed using Scaffold in the pSS patient group and the controls Out of all the aforementioned proteins in the study, LCN2 was found to be upregulated in both stimu-lated whole saliva and tear fluid in patients with pSS Low tear fluid volumes were collected individually from each patient with pSS, leading to fewer than 100 proteins identified in most of the samples in which EVs were iso-lated individually from the tear fluid of each participant However, on proteomic analysis of EVs extracted from the pooled tear sample combined from 11 patients with pSS, CPNE1 and CALM were expressed more in the patient group Moreover, the DAVID analysis of the pooled tear sample revealed cellular pathways involved in calcium-dependent phospholipid binding, and TNF-α receptor signalling, both of which are components of the adaptive immune response and comparable to the calcium ion binding pathways identified in whole saliva (Fig 1)

Discussion

By studying the proteome of biological fluids through LC-MS approaches, and possibly combined with the

Fig 1 Database for Annotation, Visualization and Integrated Discovery (DAVID) analysis delineating cellular pathways that involve proteins identified in whole saliva, tear fluid, and extracellular vesicles (EVs) Cellular pathways involving innate and adaptive immune responses, cellular component assembly, metabolism and protein folding were identified using DAVID (v 6.7, https://david.ncifcrf.gov) analysis for each sample of whole saliva, tear fluid and EVs

chromatographic separation of extracellular vesicles from

other biomolecules, the search for potential biomarkers

and therapeutic targets can be realised for SS Such

bio-markers can in turn be implemented as potential

non-invasive diagnostic tools that can be applied for monitor-ing disease progression The majority of proteomic studies

of SS have been based on saliva as the biological fluid, using different mass spectrometry approaches, in addition

Fig 2 Protein-protein interaction networks of upregulated proteins associated with primary Sjögren ’s syndrome identified in stimulated whole saliva Two distinct protein-protein interaction networks are visualised One is involved in metabolism and redox reactions, while the other plays a central role in both innate and adaptive immunity and contains the most upregulated protein in the patient group, namely neutrophil gelatinase-associated lipocalin (LCN2) The five most upregulated proteins in the patient group (Table 3) are indicated with red circles The Search Tool for the Retrieval of Interacting Genes/Proteins (http://string-db.org/) was used to generate the networks, where potential interactions of proteins with medium confidence are shown The different clusters are indicated by the same colour The colour of the connecting lines indicates the type of evidence used in predicting the associations (red gene fusion, yellow text-mining extracted from literature, purple protein-protein interaction datasets, light blue protein interaction groups, black linked across species) CALM calmodulin, CALML5 calmodulin-like protein 5, GRN granulin adipocyte plasma, APMAP

membrane-associated protein, GNA13 guanine nucleotide-binding protein subunit alpha-13, WDR1 WD repeat-containing protein 1,

SIRPA tyrosine-protein phosphatase non-receptor type substrate 1, LSP1 lymphocyte-specific protein 1

to genomics [14, 16–21] This confirms that saliva not

only reflects the salivary gland involvement that

charac-terises the disease process in SS [18, 24, 25], but

addition-ally has the potential to communicate an individual’s

current health [26, 27]

So far tear fluid has only been used to identify potential

biomarkers for pSS in a limited number of proteomic

studies [22, 23] Hence, in the current study we

hypothe-sised that by applying LC-MS alone, and in combination

with size-exclusion chromatography of both stimulated

whole saliva and tear fluid collected from patients with

pSS and from healthy controls, novel biomarkers

encom-passing both salivary and lacrimal disease target organs

could possibly be identified

In order to delineate cellular pathways involving the

up-regulated proteins identified with LC-MS in the samples

from the patients with pSS, GO and Kyoto Encyclopedia

of Genes and Genomes (KEGG) pathway

overrepresenta-tion analyses using DAVID were performed Our results

demonstrated pathways of the adaptive immune response

in the whole saliva, of the cellular component assembly in

the EVs extracted from whole saliva, of metabolism and

protein folding in the tear fluid of patients with pSS and

finally, components of the adaptive immune response in

the EVs isolated from the pooled sample of tear fluid from

patients with pSS, which was comparable to the calcium

ion binding pathways identified in whole saliva (Fig 1)

Viewed as a whole, the identified cellular pathways and

components clearly indicate the involvement of

auto-immune reactions and over-activation of the adaptive and

innate immune systems in patients with pSS, both as a

consequence of disease pathogenesis and probably also as

part of the healing process

The LC-MS analyses indicate upregulation of proteins

involved in innate immunity, cell signalling and wound

repair in whole saliva from patients with pSS

Interest-ingly, LCN2, the most upregulated protein in whole

sal-iva from patients with pSS, is an iron-binding protein

involved in the innate immune system, and is

particu-larly responsible for the activation of neutrophils [50]

This suggests the involvement of viral infection in SS pathogenesis A similar implication was depicted by Hu and co-workers [18], where they identified salivary prote-omic and genprote-omic biomarkers for SS showing upregula-tion of genes involved in the IFN pathway, thereby suggesting a potential role for viral infection in SS More-over, both GRN, a cytokine-like peptide that is central in inflammation due to its active role in wound repair and tissue remodelling [51], and CALML5, a calcium-binding protein that plays a central role in the differentiation of keratinocytes [52], were also upregulated in our patient group This finding in turn provides evidence of acinar damage and oral environment alteration

Both Giusti et al [13] and Fleissig et al [19] identified biomarkers that might include specific indication of tissue damage (e.g actin), inflammation (e.g calgranulins), and tissue repair (e.g keratin 6 L) in unstimulated whole saliva The present study identified similar potential with GRN and CALML5 in stimulated whole saliva (Table 3, Additional file 2: Figure S1) Furthermore, CALM, a calcium-binding protein that plays a role in intracellular signalling, and ESP1, a cholesterol transporter involved in cholesterol homeostasis within the endosome and/or lyso-some, were also upregulated in whole saliva from patients with pSS Similarly, previous proteomic studies on whole saliva have determined broad and distinct protein patterns that are characteristic of SS, including secretory proteins, enzymes, highly abundant immune system-related mole-cules (e.g β2-microglobulin), and cytokines such as IL-4 and IL-5 [21, 28, 29] The current STRING analysis of whole saliva also strengthens the concept of involvement and over-activation of the innate and adaptive immune sys-tem in SS This is presumably due to the upregulation of LCN2 and other related pro-inflammatory-related proteins

in the patients with pSS to form protein-protein network interactions (Fig 2)

Using size exclusion chromatography on whole saliva to isolate EVs followed by LC-MS allowed us to identify poten-tial biomarkers that are vital for activation of the innate im-mune system and adipocyte differentiation More precisely,

Table 3 Highly upregulated proteins in stimulated whole saliva from patients with pSS

Number Gene Related proteina Replicates (pSS

: C)

Spectral counts (pSS : C)

Classification and functionb

1 LCN2 Neutrophil gelatinase-associated

lipocalin

10 : 2 38 : 2 Iron-binding protein; innate immunity (neutrophils)

2 GRN Granulins 7 : 0 12 : 0 Cytokine-like peptide; inflammation, wound repair, tissue

remodelling

3 CALM Calmodulin 7 : 1 17 : 1 Calcium-binding protein; intracellular signalling

4 NPC2 Epididymal secretory protein 1 6 : 0 11 : 0 Cholesterol transporter; cholesterol homeostasis

(endosome/lysosome)

5 CALML5 Calmodulin-like protein 5 5 : 0 13 : 0 Calcium-binding protein; differentiation of keratinocytes

a

The five most upregulated proteins in whole saliva from patients with primary Sjögren ’s syndrome (pSS) deviating in replicates, i.e number of individuals (frequency), and spectral counts, as identified by proteomics analysis and Scaffold (v 4.4.6, http://www.proteomesoftware.com/products/scaffold/ ) b

The classification and functions of the proteins presented were identified using publicly available databases, such as UniProt ( http://www.uniprot.org ) C controls

the most upregulated protein in EVs from whole saliva,

namely APMAP, is an enzyme central to adipocyte

develop-ment We have recently shown increased occurrence of

adipose tissue replacement in minor salivary gland biopsies

from patients with SS [53] Interestingly, these adipocytes

were detected in areas rich in IL-6, suggesting their active

involvement in immune reactions Hence, the upregulation

of APMAP in stimulated whole saliva could be an indication

of adipocyte involvement in disease progression

More-over, both GNA13, a G-protein that consequently plays

a role in transmembrane signalling, and WDR1, a

regulatory protein involved in the disassembly of actin filaments, are proteins needed to drive inflammation and tissue damage, respectively

Interestingly, SIRPA is another potential biomarker of the pro-inflammatory process, as it regulates NK cells and dendritic cells Furthermore, LSP1, being involved in innate immunity, specifically neutrophil activation and chemotaxis, is another possible indicator of the involve-ment of viral infection in the pathogenesis of SS (Table 4, Additional file 3: Figure S2) One major protein-protein interacting network was identified for EVs isolated from

Fig 3 Protein-protein interaction networks of upregulated proteins associated with primary Sjögren ’s syndrome detected in extracellular vesicles from whole saliva One major protein-protein interaction network is visualised The proteins identified are involved in the cytoskeleton, in addition to cell migration and cell junction Out of the five most upregulated proteins in pSS (Table 4), indicated with red circles, both guanine nucleotide-binding protein subunit alpha-13 (GNA13) and WD repeat-containing protein 1 (WDR1) are present within this protein network The Search Tool for the Retrieval of Interacting Genes/Proteins (http://string-db.org/) was used to generate the networks, where potential interactions of proteins with medium confidence are shown The different clusters are indicated by the same colour The colour of the connecting lines indicates the type of evidence used in predicting the associations (red gene fusion, yellow text-mining extracted from literature, purple protein-protein interaction datasets, light blue protein interaction groups, black linked across species) APMAP membrane-associated protein, SIRPA tyrosine-protein phosphatase non-receptor type substrate 1, LSP1 lymphocyte-specific protein 1